Catharanthus roseus produces a wealth of biologically active alkaloids including the potent anticancer molecules vincristine and vinblastine and the antihypertension agent ajmalicine. The complex nature of the C. roseus biosynthetic pathway leads to a wide variety of valuable compounds, however, the diversity of the pathway also results in small quantities of the desired materials. A solution to the impractical amounts of medicinal compounds produced by Catharanthus roseus exists in the elucidation of the largely unknown biosynthetic pathway. The alkaloids of interest all derive from the common precursor strictosidine which is deglycosylated to form several intermediates existing in equilibrium. These-intermediates lead to one of four pathways which produce the biologically active alkaloids. This proposal focuses on the enzymes of the three less familiar pathways leading to the alkaloids ajmalicine, isositsirikine, and yohimbine. The information I obtain will ultimately aid in the reengineering of the pathway to suppress production of less useful alkaloids, thereby increasing formation of more valuable alkaloids such as the chemotherapeutics vinblastine and vincristine. I will isolate and identify at least one enzyme which catalyzes one of several of the major channels of the C. roseus biosynthetic pathway using two aims. Aim one will focus on the synthetic modification of strictosidine and tryptamine to incorporate a series of labels, for crosslinking enzymes, and tags, for detecting, into a single molecule. The labels will range from photolabile aryl azides to diazirines. The tags will include radiolabels as well as alkynes and ketones, capable of incorporating biotin labels through click chemistry and oxime formation, respectively. I will form the molecules through a combination of synthetic and chemoenzymatic reactions. The strictosidine analogs will then be assessed for enzymatic recognition in cell free extracts. The labeled and tagged analogs that are recognized by the C. roseus enzymes will be crosslinked to the proteins in cell extracts. Following isolation by 2-D electrophoresis, I will detect the labeled enzymes by radiography, in the case of radiolabeled tags, and western blot analysis when dealing with biotin tagged molecules. The Danforth Plant Center will sequence the proteins and experienced members of the lab will compare these sequences to a cDNA library. PUBLIC HEALTH RELEVANCE: The knowledge gained during my research will allow for reengineering of the pathways of C. roseus to increase production of medicinal compounds and facilitate access to practical quantities of these therapies. This information could ultimately lead to more effective and more affordable treatments of disease.